Methods and systems for producing products using engineered sulfur oxidizing bacteria

ABSTRACT

Methods and systems for producing a biofuel using genetically modified sulfur-oxidizing and iron-reducing bacteria (SOIRB) are disclosed. In some embodiments, the methods include the following: providing a SOIRB that have been genetically modified to include a particular metabolic pathway to enable them to generate a biofuel; feeding a first source of ferric iron to the SOIRB; feeding sulfur, water, and carbon dioxide to the SOIRB; producing at least the first particular biofuel, a first source of ferrous iron, sulfate, excess ferric iron, and an SOIRB biomass; electrochemically reducing the excess ferric iron to a second source of ferrous iron; providing an iron-oxidizing bacteria that have been genetically modified to include a particular metabolic pathway to enable them to generate a second biofuel; producing at least the second biofuel, a second source of ferric iron, and an IOB biomass; and feeding the second source of ferric iron to the SOIRB.

CROSS REFERENCE TO RELATED APPLICATION(S)

This application is a continuation of U.S. Non-provisional PatentApplication No. 14/377,223, filed Aug. 7, 2014, which claims the benefitof U.S. Provisional Application No. 61/599,550, filed Feb. 16, 2012,each of which is incorporated by reference as if disclosed herein intheir entirety.

BACKGROUND

There has been interest in the development of liquid biofuels as theseprocesses have the potential to directly fix carbon dioxide intotransportation fuels, which is potentially carbon neutral andpolitically attractive. Cellulose based biofuels including bioethanol,algae-derived lipids, cyanobacteria, and algae derived hydrogen (H₂) areamong the most studied biofuels. Despite the promise of thesetechnologies and processes, there are specific limitations that precludetheir wide-spread application. For example, post-processing of algalcells and derived lipids imposes higher production costs on algalbiodiesel. The production rates of H₂ from cyanobacteria still remainslow and productivity needs to be improved. Genetically engineeredphotosynthetic organisms have also been explored for bioethanolproduction. However separation of ethanol from the aqueous phase remainsa challenge.

Microbial fuel cells have been under investigation and development formore than a century, as the use of cells to harvest electrical energyfrom waste streams is attractive for many reasons. In a biofuel cell,biological catalysts are used on an anode to oxidize biofuels, and acathode is created that can use the generated electrons to reduce oxygento water. These systems can either be microbial with living cells on theelectrodes, or they can be enzymatic systems, with purified enzymes onthe electrodes. In both designs, power can be generated from theoxidation of biofuels, and there are many advantages to these systemsover conventional fuel cells and other power generation schemes.However, much research still needs to be done with microbial fuel cellsto make them practical and cost-efficient. A significant limitation forboth enzymatic and microbial fuel cells is the need for mediators toenable electrical contact between the biological components andinorganic electrode. In some microbial systems, these mediators are madeby the organisms themselves, and in other technologies, syntheticmediators are added to the system. In some systems, cells must makephysical contact with the electrodes for electron transfer. This can bea significant limitation as it reduces the cellular mass that can beused for biochemical conversion.

Natural gas is frequently purified from “sour gas”, which is a naturalgas deposit containing high volumes of sulfur or hydrogen sulfide. Theseparate sulfur compounds are a significant waste product produced bythese processes, lacking an economical or sustainable disposal process.

SUMMARY

Living cells have the ability to reproduce and maintain their catalyticmachinery, and their metabolic pathways can be rationally altered tomeet desired process objectives. But, efficient electron transfer fromthe electrode to the organism can limit metabolic production, and theuse of mediating species can result in a process that is noteconomically viable. One way to address these limitations is to explorealternative organisms that naturally utilize mediators that are moreattractive. The disclosed subject matter includes the metabolicengineering of chemolitotrophic sulfur-oxidizing and iron-reducingbacteria (SOIRB), such as Acidithiobacillus ferrooxidans, to develop aprocess that can overcome these limitations. SOIRB have the naturalability to fix carbon dioxide while oxidizing sulfur and reducing ferriciron (Fe³⁺) to ferrous iron (Fe²⁺).

Aspects of the disclosed subject matter include the use of engineeredstrains of the SOIRB, e.g., A. ferrooxidans, for the production ofbiofuels. SOIRB fix carbon dioxide for cell-synthesis while derivingenergy from the oxidation of sulfur and reduction of ferric iron toferrous iron. The ferrous iron produced upon reduction of ferric ironcan be electrochemically oxidized back to ferric iron in anelectrochemical reactor, and additional ferric iron can be added fromany ferric iron-rich stream. In this way, the SOIRB can be grownefficiently in a bioreactor using ferric iron as the mediator.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings show embodiments of the disclosed subject matter for thepurpose of illustrating the invention. However, it should be understoodthat the present application is not limited to the precise arrangementsand instrumentalities shown in the drawings, wherein:

FIG. 1 a schematic diagram of systems according to some embodiments ofthe disclosed subject matter;

FIG. 2 a schematic diagram of systems according to some embodiments ofthe disclosed subject matter;

FIG. 3 is a chart of a method according to some embodiments of thedisclosed subject matter;

FIG. 4 is a diagram showing production of isobutanol via an oxidizingbacteria having a modified genetic sequence according to someembodiments of the disclosed subject matter; and

FIG. 5 is a chart of a method according to some embodiments of thedisclosed subject matter.

DETAILED DESCRIPTION

Referring now to FIGS. 1 and 2, aspects of the disclosed subject matterinclude methods and systems that include the application ofchemolitotrophic sulfur oxidizing bacteria for concomitant carbondioxide fixation, conversion of the carbon dioxide to a biofuel such asisobutanol, and oxidation of elemental sulfur to sulfate compounds. Insome embodiments, ferric iron is simultaneously reduced back to ferrousiron and excess ferric iron is electrochemically reduced. Additionalferric iron can be added from other ferric iron-rich sources. Metabolicengineering is used to introduce a new pathway into the bacteria thatstarts with the precursors for amino acid synthesis to create butanols,e.g., isobutanol, etc.

Some embodiments include systems and methods for producing products suchas biofuels and chemicals. As shown in FIG. 1, some embodiments includea system 100 for producing biofuels using genetically modifiedsulfur-oxidizing and iron-reducing bacteria (SOIRB) 102 grown in a firstbioreactor 104 that are fed elemental sulfur, ferric iron, and carbondioxide. The elemental sulfur is oxidized to sulfate providing electronsto the SOIRB, the carbon dioxide is used as a base material to be fixedinto a biofuel or chemical, and the ferric iron functions as theultimate electron acceptor and is reduced to ferrous iron. Initially,the ferric iron is typically provided from a first source 106 that isexternal to system 100, e.g., a ferric iron-rich stream in fluidcommunication with bioreactor 104. Typically, but not always, the ferriciron used in system 100 is substantially provided by a second source 108that is generated in a second bioreactor 110. Second source 108 offerric iron serves as a mediator for transferring electrons from SOIRB102. In some embodiments, substantially all of the ferric iron used bybioreactor 104 is provided by a source external to system 100.

Bioreactor 104 includes SOIRB 102 that have been genetically modified toinclude a particular metabolic pathway to enable them to generate abiofuel 112. The operating parameters of bioreactor 104 are typicallyoptimized to maximize the production of ferrous iron 114. Ferrous iron114 is introduced to bioreactor 110. In some embodiments, bioreactor 104will be configured so as to be fed about 70 mM to about 500 mM ferriciron. In some embodiments, the pH will likely be maintained in the rangeof about 1.5 to 4 and temperature at about 20 to 40 degrees Celsius.Methods and systems according to the disclosed subject matter haveoperating conditions that are optimized for optimal yield, conversion,etc. Bioreactors included in methods and systems according to thedisclosed subject matter are typically operated in a continuous flowmode to maximize the conversion of the substrates to the products.

Excess ferric iron 115, which remains in bioreactor 104, is introducedto an electrochemical reactor 116, which is in fluid communication withthe bioreactor. Electrochemical reactor 116 includes electrodes, i.e.,an anode 117 and a cathode 118, a separator 120, and source ofelectrical energy 121. In some embodiments, cathode 118 is formedsubstantially from carbon and anode 117 is formed substantially fromcarbon and/or other known materials. In some embodiments, flow throughor flow by porous electrodes are used. In some embodiments, separator120 is a cation selective membrane, to allow for proton transfer acrossthe membrane to achieve acid balance.

Electrochemical reactor 116 is typically configured to electrochemicallyreduce ferric iron 115 to a second source 122 of ferrous iron usingsource of electrical energy 121. In system 100, ferrous iron 114 will becontinually regenerated back to ferric iron, i.e., second source 108,and the recycle loop can be theoretically closed without the need foradditional ferric iron input from first source 106 beyond startup.

Some embodiments of the disclosed subject matter include systems havingholding tanks for the ferrous iron rich streams and ferric iron richstreams to enable the electrochemical production of ferrous iron tooperate independently of the bioreactor to take advantage of thetransient pricing and availability of electricity. For example, at timesduring the day when electricity is least expensive, the electrochemicalsystem would produce as much ferrous iron as possible to be stored andused slowly by the bioreactor, which will be operating continuously.This solves a major limitation encountered in photo bioreactors whereinterruptions in light can negatively impact the process.

System 100 includes a source of water 123, a source 124 of carbondioxide, a source of oxygen 125, and a source of elemental sulfur126—all except oxygen are in fluid communication with bioreactor 104 andall except sulfur are in fluid communication with bioreactor 110. Insome embodiments, source 124 is carbon dioxide removed from air orenergy plant emissions. In some embodiments, either in place of or inaddition to carbon dioxide, carbonate, e.g., from mineral sources, isfed to bioreactor 104.

Second bioreactor 110 includes iron-oxidizing bacteria (IOB) 128 thathave been genetically modified to include a particular metabolic pathwayto enable them to generate a second particular biofuel (not shown) orbiofuel 112. First and second sources 114, 122 of ferrous iron are influid communication with bioreactor 110. In some embodiments, a thirdsource 132 of ferrous iron is also in fluid communication withbioreactor 110.

Referring now to FIG. 2, some embodiments include a system 100′ that donot include second bioreactor 110. Instead, ferrous iron 114 produced inbioreactor 104 is directed to a side of electrochemical reactor 116 thatincludes anode 117 where it is oxidized to produce a second source orferric iron 108′, which is then recycled back to the bioreactor and fedto SOIRB 102.

Although not included in FIGS. 1 and 2, in some embodiments, systems 100and 100′ includes pumps for pumping the various constituents, into,through, and out of the system. In addition, the pumps are typicallyprogrammable to allow electrochemical reactor 116 to be turned off whenthe price of electricity is high and turned on when the price is low.Also, the pumps typically include a separator unit to separate one ormore particular constituents that are to be pumped to other componentsof system 100 from the other constituents.

Referring now to FIGS. 3 and 4, some embodiments include a method 200for producing a biofuel using genetically modified sulfur-oxidizing andiron-reducing bacteria (SOIRB). As shown in FIG. 3, at 202, SOIRB thathave been genetically modified to oxidize sulfur and reduce iron areprovided.

As shown best in FIG. 4, in some embodiments, the SOIRB is substantiallyA. ferrooxidans, e.g., wild type A. ferrooxidans 23270 strain orsimilar, and the IOB are genetically modified by including at least oneof a 2-keto-acid decarboxylase gene (outlined by box) and an alcoholdehydrogenase gene or similar. The production of isobutanol inprokaryotic hosts begins with the amino acid biosynthesis pathways.These pathways produce 2-keto acids, and these are converted toaldehydes using a 2-keto-acid decarboxylase. Alcohol dehydrogenase isthen used to convert the aldehydes to alcohols.

In the case of isobutanol, the valine biosynthesis pathway is used, andthe starting precursor is 2-keto-isovalerate.

In some embodiments, the SOIRB provided are genetically modified to beable to utilize hydrogen as an electron donor. The use of hydrogen as amediator improves system efficiency because hydrogen may be cogeneratedwith ferrous iron during the electrochemical regeneration step. Thereare various hydrogenase enzymes from different organisms that can beused in microbial biohydrogen production. But other hydrogenase enzymes,found in organisms such as Metallosphaera sedula and hodopseudomonaspalustris, enable hydrogen uptake and its use as a reductant. Of course,because A. ferrooxidans also contains its own hyddrogenase that can beused as described here, in some embodiments, the SOIRB provided are notgenetically modified to be able to utilize hydrogen as an electrondonor.

Referring again to FIG. 3, at 204, a first source of ferric iron is fedto the SOIRB. At 205, water is fed to the IOB. At 206, carbon dioxide isfed to the IOB. At 207, elemental sulfur is fed to the SOIRB. At 208,ferrous iron, sulfate compounds, excess ferric iron, and an SOIRBbiomass are produced. In some embodiments, ferrous iron production ismaximized during 208. During 208, elemental sulfur is oxidized andferric iron is reduced. At 210, the excess ferric iron remaining iselectrochemically reduced to a second source of ferrous iron. Hydrogenis also often produced while electrochemically reducing the ferric iron.Next, at 212, iron-oxidizing bacteria (IOB) that have been geneticallymodified to include a particular metabolic pathway to enable them togenerate a second particular biofuel are provided. Still at 212, thesecond source of ferrous iron and the hydrogen are fed to the IOB. Thesecond source of ferrous iron serves as a mediator for transferringelectrons to the IOB. At 214, water, carbon dioxide, the first source offerrous iron, and oxygen are fed to the IOB. At 216, a particularbiofuel, a second source of ferric iron, and an IOB biomass areproduced. In some embodiments, the biofuel is one of isobutanol, a longchain alcohol, or an alkane. At 218, the second source of ferric iron isfed to the SOIRB and the process returns to 208 where additionalbiofuel, ferrous iron, sulfate compounds, excess ferric iron, and anSOIRB biomass are produced.

Referring now to FIG. 5, some embodiments include a method 400 forproducing a chemical or biofuel using genetically modifiedsulfur-oxidizing bacteria (SOB). At 402, SOB that have been geneticallymodified to include a particular metabolic pathway to enable them togenerate a first particular biofuel or chemical are provided. At 404,elemental sulfur is fed to the SOB. At 406, water is fed to the SOB. At408, carbon dioxide is fed to the SOB. At 410, oxygen is fed to the SOB.At 412, a biofuel or chemical, sulfate, and an SOB biomass are produced.In some embodiments, the chemical is one of a commodity chemical, aspecialty chemical, a feedstock such as an acid, an amino acid, acarbohydrate, or a combination thereof. At 412, elemental sulfur isoxidized.

Reverse microbial fuel cells according to the disclosed subject matterutilize carbon dioxide and electrical input to produce infrastructurecompatible transportation fuels. The technology uses cultures of IOB,e.g., A. ferrooxidans, which are genetically modified to produceisobutanol.

Systems and methods according to the disclosed subject matter use onlyabundant, inexpensive redox mediators. They do not use costly rare earthelements or organic redox shuttles, and thus can be potentially deployedeconomically at scale. They potentially exceed an overall efficiencygreater than one percent and butanol has desirable fuel properties andis compatible with transportation-fuel infrastructure.

Although the disclosed subject matter has been described and illustratedwith respect to embodiments thereof, it should be understood by thoseskilled in the art that features of the disclosed embodiments can becombined, rearranged, etc., to produce additional embodiments within thescope of the invention, and that various other changes, omissions, andadditions may be made therein and thereto, without parting from thespirit and scope of the present invention.

What is claimed is:
 1. A method for producing a biofuel usinggenetically modified bacteria, said method comprising: providing in afirst bioreactor bacteria that is genetically modified to oxidizeelemental sulfur and reduce iron (SOIRB), wherein said SOIRB includes avaline biosynthesis metabolic pathway that produces 2-keto acids and isgenetically modified to include a 2-keto-acid decarboxylase gene and analcohol dehydrogenase gene, said SOIRB being genetically modified sothat said genes in said SOIRB convert said 2-keto acids to isobutanol;feeding a source of ferric iron to said SOIRB; feeding sulfur, water,and carbon dioxide to said SOIRB; oxidizing said sulfur; reducing saidfirst source of ferric iron; producing at said first bioreactor at leastone of biofuel, a source of ferrous iron, sulfate, excess ferric iron,and an SOIRB biomass; providing in a second bioreactor an iron-oxidizingbacteria (IOB), said IOB includes a valine biosynthesis metabolicpathway that produces 2-keto acids and is genetically modified toinclude a 2-keto-acid decarboxylase gene and an alcohol dehydrogenasegene, said IOB being genetically modified so that said genes in said IOBconvert said 2-keto acids to isobutanol; and feeding said source offerrous iron to said second bioreactor.
 2. The method according to claim1, further comprising: feeding water, carbon dioxide, and oxygen to saidIOB; producing at said second bioreactor at least one of said secondbiofuel, a second source of ferric iron, and an IOB biomass; and feedingsaid second source of ferric iron to said SOIRB.
 3. The method accordingto claim 2, further comprising: electrochemically reducing said excessferric iron to a second source of ferrous iron; and feeding said secondsource of ferrous iron to said IOB.
 4. The method according to claim 2,wherein said first or second biofuel is one of isobutanol, a long chainalcohol, and an alkane.
 5. The method according to claim 1, wherein saidSOIRB are genetically modified to be able to utilize hydrogen as anelectron donor.
 6. The method according to claim 1, further comprising:feeding a third source of ferrous iron to said IOB.
 7. The methodaccording to claim 1, wherein said SOIRB is Acidithiobacillusferrooxidans.
 8. The method according to claim 1, wherein said IOB isAcidithiobacillus ferrooxidans.
 9. A system for producing biofuels usinggenetically modified sulfur-oxidizing and iron-reducing bacteria, saidsystem comprising: a first bioreactor including sulfur-oxidizing andiron-reducing bacteria (SOIRB), wherein said SOIRB includes a valinebiosynthesis metabolic pathway that produces 2-keto acids and isgenetically modified to include a 2-keto-acid decarboxylase gene and analcohol dehydrogenase gene, said SOIRB being genetically modified sothat said genes in said SOIRB convert said 2-keto acids to isobutanol; afirst source of ferric iron in fluid communication with said firstbioreactor; a first source of ferrous iron produced by said firstbioreactor, said first bioreactor configured to feed said first sourceof ferrous iron to a second bioreactor, said second bioreactor includingiron oxidizing bacteria (IOB), wherein said IOB includes a valinebiosynthesis metabolic pathway that produces 2-keto acids and isgenetically modified to include a 2-keto-acid decarboxylase gene and analcohol dehydrogenase gene, said IOB being genetically modified so thatsaid genes in said IOB convert said 2-keto acids to isobutanol; and asecond source of ferric iron produced by said second bioreactor, saidsecond bioreactor configured to feed said second source of ferric ironto said first bioreactor.
 10. The system according to claim 9, anelectrochemical reactor in fluid communication with said firstbioreactor and said second bioreactor, said electrochemical reactorconfigured to directly reduce excess ferric iron provided by said firstbioreactor to a second source of ferrous iron and to generate a streamincluding said second source of ferrous iron to be fed to said secondbioreactor.
 11. The system according to claim 10, wherein saidelectrochemical reactor includes a cathode formed substantially from atleast one of nickel and glassy carbon.
 12. The system according to claim9, further comprising: a source of water in fluid communication withsaid first bioreactor; a source of sulfur in fluid communication withsaid first bioreactor; and a source of carbon dioxide in fluidcommunication with said first bioreactor; a source of water in fluidcommunication with said second bioreactor; a source of oxygen in fluidcommunication with said second bioreactor; a source of carbon dioxide influid communication with said second bioreactor; and at least a thirdsource of ferrous iron in fluid communication with said secondbioreactor.
 13. The system according to claim 9, wherein said SOIRB aregenetically modified to be able to utilize hydrogen as an electrondonor.
 14. The system according to claim 9, wherein said SOIRB isAcidithiobacillus ferrooxidans.
 15. The system according to claim 9,wherein said IOB is Acidithiobacillus ferrooxidans.
 16. A method forproducing a chemical or biofuel using genetically modifiedsulfur-oxidizing (SOB), said method comprising: providing a SOB thathave been genetically modified to include a particular metabolic pathwayto enable them to generate a first particular chemical or biofuel;feeding sulfur, water, carbon dioxide, and oxygen to said SOB; oxidizingsaid sulfur; and producing at least said chemical or biofuel, sulfate,and an SOB biomass.
 17. The method according to claim 14, wherein saidbiofuel is one of isobutanol, a long chain alcohol, or an alkane. 18.The method according to claim 14, wherein said chemical is one of acommodity chemical, a specialty chemical, a feedstock such as an acid,an amino acid, a carbohydrate, and a combination thereof.
 19. The methodaccording to claim 14, wherein said SOB is substantiallyAcidithiobacillus ferrooxidans.
 20. The method according to claim 14,wherein said SOB are genetically modified to be able to utilize hydrogenas an electron donor.